Ann. N.Y. Acad. Sci. 1044: 51–59 (2005). © 2005 New York Academy of Sciences.
Genetic and Functional Characterization of
Isolated Stromal Cell Lines from the
KATJA C. WEISELa AND MALCOLM A. S. MOOREb
aUniversity of Tübingen, Medical Center,
Department of Hematology, Oncology, and Immunology, Tübingen, Germany
bLaboratory of Developmental Hematopoiesis, Memorial Sloan-Kettering Cancer Center,
New York, New York, USA
ABSTRACT: The hematopoietic system interacts with a supportive stromal
environment allowing maintenance and differentiation of hematopoietic stem
cells (HSCs). The aorta-gonado-mesonephros (AGM) region serves as a unique
embryonic microenvironment, generating the first adult repopulating HSCs in
the mouse embryo. To eludicate factors involved in hematopoietic support and
induction of hematopoietic differentiation, we isolated more than 100 stromal
cell clones derived from the AGM region of embryonic day (E) 10.5 mouse
embryos for functional and genetic analysis. Selected isolated AGM stromal
cell lines are highly efficient in supporting maintenance and expansion of
mouse and human hematopoietic stem and progenitor cells. In addition, we can
demonstrate for the first time that AGM stromal cell lines are also potent
inducers of hematopoietic differentiation of murine embryonic stem cells.
Stromal gene array analysis has identified genes that could play a role in
KEYWORDS: aorta-gonado-mesonephros region; stromal coculture; murine ES
cells; ex vivo expansion
During hematopoietic development, hematopoiesis first begins in the yolk sac
(YS) at day 7.5 postgestation (E7.5) before shifting into the fetal liver, spleen, and
bone marrow.1,2 Nondefinitive progenitors first appear in YS blood islands at E8.5.3
Definitive murine hematopoietic stem cells (HSCs) emerge from the intraembryonic
aorta-gonado-mesonephros (AGM) region, appearing initially on the floor of the
dorsal aorta.4,5 These hematopoietic cells, capable of reconstituting definitive, adult
hematopoiesis, first appear at day 10.5 after gestation.
Address for correspondence: Katja C. Weisel, M.D., University of Tübingen, Department of
Hematology, Oncology, and Immunology, Otfried-Müller-Strasse 10, 72076 Tübingen, Germany.
Voice: +49-7071-2982726; fax: +49-7071-293671.
52 ANNALS NEW YORK ACADEMY OF SCIENCES
Stromal cells are known to be essential in the regulation of hematopoiesis. In non-
cytokine conditions, hematopoiesis is not maintained when stromal cell growth is
impeded.6 Further studies have demonstrated that bone marrow (BM) and fetal liver
stromal cells support long-term proliferation of HSCs, as measured by late second-
ary progenitor colony formation and cobblestone area formation beneath the
stroma.7–9 A number of immortalized stromal cell lines from adult murine BM and
fetal liver have been developed for long-term support of both murine and human
HSCs.8–10 More recently, the presumptively unique characteristics of the embryonic
hematopoietic microenvironment have been investigated further, and a limited num-
ber of stromal cell lines from the YS and the AGM region have been developed that
support murine and human hematopoiesis.11–16 However, until now, only two cell
lines derived from the AGM region and the surrounding urogenital ridge have been
reported that are able to support human hematopoiesis in a cytokine-independent
manner. More recently, BM-derived stromal cell lines have been reported that can
induce hematopoietic differentiation when cocultured with totipotent embryonic
stem (ES) cells. For hematopoietic differentiation of murine17 and human18 ES cells,
so far the OP9 stromal cell line derived from the BM of the op−/− mouse is well
established as a potent inducer of hematopoiesis.
Murine mesenchymal stromal lines supporting growth and maintenance of
hematopoietic stem and progenitor cells derived from marrow, spleen, and fetal liver
represent several stages along the vascular smooth muscle cell (VSMC) differenti-
ation pathway.19,20 However, until now, mechanisms of hematopoietic differentia-
tion and hematopoietic stem cell maintenance or expansion have not been fully
Here, we describe the isolation and characterization of more than 100 cloned stro-
mal cell lines derived from the AGM region of E10.5 mouse embryos. When cocul-
tured with the stromal cells, hematopoietic cells, especially primitive hematopoietic
stem/progenitor cells in adult mouse bone marrow and human cord blood, prolifer-
ated significantly without additional cytokines. Moreover, one of these AGM strom-
al cell lines induced significant hematopoietic differentiation of murine ES cell lines
comparable to that reported in OP9 stromal coculture. Genetic characterization of
supporting lines, including microarray analysis, revealed a mesenchymal and vascu-
lar smooth muscle cell phenotype.
Functional Characterization of Isolated AGM Stromal Cell Lines
Effect of AGM Stromal Cell Lines on Mouse BM Progenitor Cells
A total of 106 stromal cell lines (AGM-S1 to AGM-S106) were obtained from
E10.5 mouse embryos by dissecting the AGM region and culturing outgrowing stro-
mal cells as described.13 It has previously been shown that AGM-derived stromal
cell lines are capable of supporting murine adult hematopoiesis.13,14 As a first
screening to determine whether the generated clones could support growth and
maintenance of HSCs, we examined the ability of the 106 established stromal cell
lines to support hematopoiesis following coculture with mouse bone marrow mono-
59WEISEL & MOORE: GENETIC AND FUNCTIONAL CHARACTERIZATION
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